Search

Several in vitro cellular models have been developed with the aim to reproduce and dissect human granulomatous responses, the hallmark of tuberculosis (TB) immunopathogenesis. In that context, we compared two- (2D) versus three-dimensional (3D) granuloma models resulting from infection of human peripheral blood mononuclear cells with M. tuberculosis (Mtb) in the absence or presence of a collagen-based extracellular matrix (ECM). Granuloma formation was found to be significantly enhanced in the 2D model. This feature was associated with an earlier chemokine production and lymphocyte activation, but also a significantly increased bacterial burden. Remarkably, the reduction in Mtb burden in the 3D model correlated with an increase in GM-CSF production. GM-CSF, which is known to promote macrophage survival, was found to be inherently induced by the ECM. We observed that only 3D in vitro granulomas led to the accumulation of lipid inclusions within Mtb. Our data suggest that a hypoxic environment within the ECM could be responsible for this dormant-like Mtb phenotype. Furthermore, exposure to a TNF-α antagonist reverted Mtb dormancy, thereby mimicking the reactivation of TB observed in rheumatic patients receiving this therapy. To conclude, we showed that only in vitro granulomas generated in the presence of an ECM could recapitulate some clinically relevant features of granulomatous responses in TB. As such, this model constitutes a highly valuable tool to study the interplay between immunity and Mtb stress responses as well as to evaluate novel treatment strategies.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies., PLoS Pathogens, 16 (4), ISSN:1553-7374, ISSN:1553-7366

Background: Most biotherapeutics are glycoproteins and glycosylation is important for their function. Concerns regarding immunogenicity arise from the high heterogeneity in glycosylation that is difficult to control and can deviate from human glycosylation if produced in non-human cell lines. Some of this human-like glycosylation may constitute signatures that allows entry into and activation of immune cells via recognition by receptors with carbohydrate ligand specificity (lectin receptors). The goal of my work is to investigate whether and how glycosylation affects immune recognition of therapeutic antibodies. Approach: Using the tool antibody rituximab (RTX) with a high incidence in clinical immunogenicity (i.e., anti-drug antibodies), I investigated glycosylation-mediated effects covering the three main aspects: 1. antigen recognition and uptake by dendritic cells (DCs), 2. antigen presentation to T cells and 3. humoral immune responses. Results: I found that human DCs bind and internalize unmodified RTX stronger compared to its aglycosylated form suggesting that glycosylation mediates uptake after recognition by glycan-specific receptors. In addition, I can show that DC-uptake is increased or decreased if glycosylation is selectively modified to recognize activating or inhibitory lectin receptors. Furthermore, glycosylation seems to influence antigen presentation by DCs because specific glycovariants tend to induce either stronger or weaker T cell activation. Finally, I could demonstrate that antibody glycosylation impacts humoral immune responses. Conclusion: I could show that glycosylation is relevant for immune recognition of the therapeutic antibody RTX. Defined glycan structures can modulate immune recognition subsequently leading to increased or decreased responses. Thus, glyco-engineering may be used to reduce clinical immunogenicity.

Background: Naproxen is a member of the aryl acetic acid class of non-steroidal anti-inflammatory drugs that has anti-inflammatory, antipyretic and analgesic properties. Naproxen is extensively metabolized in the liver by P450 enzymes to 6-O-desmethylnaproxen. Both parent drug and desmethyl metabolite undergo phase II biotransformation to their respective acyl glucuronide conjugated metabolites. Naproxen-induced idiosyncratic hepatotoxicity is a rare event. The mechanism of the tissue injury and the nature of the chemical entity involved in the adverse reaction have not been studied. Aims: To explore and characterise naproxen (metabolite)-specific immune responses from patients with or without liver injury. Methods: The chemical instability and protein reactivity of naproxen acyl glucuronide were investigated in vitro and an adduct with human serum albumin was generated. Peripheral blood mononuclear cells were isolated from patients and healthy volunteers. Lymphocyte proliferation and IFNγ secretion against naproxen, its major metabolites and the serum albumin adduct were examined. T-cell cloning by limiting dilution was undertaken in order to generate drug-specific T-cell clones and appropriate immune functional assays were used to characterize responses. Next generation sequencing was used in order to interrogate the transcriptional signature of peripheral blood mononuclear cells in response to naproxen and its major metabolites. Results: Naproxen acyl glucuronide is chemically unstable with an identified degradation half-life of 2.72 hrs in 0.1 M phosphate buffer at 37°C (pH 7.4). Furthermore, irreversible binding was measured at 247.37 ± 57.64 nmol of drug per μmol serum albumin under 50 : 1 metabolite : protein molar ratio incubation at 37°C (pH 7.4). 6-O-desmethylnaproxen, but not naproxen or naproxen acyl glucuronide, exhibited lymphocyte stimulation in a patient with naproxen-induced liver injury. A CD4+CD8+ Th17 desmethylnaproxen – responsive T-cell clone was identified. The clone was found to be MHC-class II restricted and become activated via a pharmacological-interaction mechanism. 6-O-desmethylnaproxen exerts significant differential gene expression in peripheral blood mononuclear cells isolated from patients and healthy volunteers and an overall down-regulatory pattern was revealed. Conclusion: Results show lymphocyte responses were directed to the primary desmethyl metabolite of naproxen, but not the parent drug or acyl glucuronide metabolite. In addition, the desmethyl metabolite shows consistent differential gene expression in human peripheral blood mononuclear cells. Taken together, this evidence suggests an overall immune-mediated basis for naproxen-induced idiosyncratic hepatotoxicity with a role for the inert oxidative metabolite. Whilst the FDA considers acyl glucuronide adduction of protein to be responsible for the liver reactions associated with carboxylic acid drugs such as naproxen, no evidence of this was found herein.